A haloperoxidase peroxygenase denoted AaP from the agaric basidiomycete strain Agrocybe aegerita (strain TM-A1) was found to oxidize aryl alcohols and aldehydes. The AaP peroxygenase was purified from A. aegerita TM A1 by several steps of ion chromatography and SDS-PAGE, the molecular weight was determined and the N-terminal 14 amino acid sequence was determined after 2-D electrophoresis but the encoding gene was not isolated (Ullrich et al., 2004, Appl. Env. Microbiol. 70(8): 4575-4581).
WO 2006/034702 A1 discloses methods for the enzymatic hydroxylation of non-activated hydrocarbons, such as, naphtalene, toluol and cyclohexane, using the AaP peroxygenase enzyme of Agrocybe aegerita TM A1. This is also described in Ullrich and Hofrichter, 2005, FEBS Letters 579: 6247-6250.
DE 103 32 065 A1 discloses methods for the enzymatic preparation of acids from alcohols through the intermediary formation of aldehydes by using the AaP peroxygenase enzyme of Agrocybe aegerita TM A1.
A method was reported for the rapid and selective spectrophotometric direct detection of aromatic hydroxylation by the AaP peroxygenase (Kluge et al., 2007, Appl Microbiol Biotechnol 75: 1473-1478).
A second peroxygenase capable of aromatic peroxygenation was isolated from the coprophilous fungus Coprinus radians and characterized, the N-terminal 16 amino acids were identified and aligned with the N-terminal 14 amino acids of the AaP enzyme of the A. aegerita strain earlier published; but the encoding gene was not isolated (Anh et al., 2007, Appl Env Microbiol 73(17): 5477-5485).
It is well-known that a direct regioselective introduction of oxygen functions (oxygenation) into organic molecules constitutes a problem in chemical synthesis. It is particularly difficult to catalyse the selective N-oxygenation of aromatic heterocycles of the pyridine type. The products, heterocyclic N-oxides, are important intermediates in a wide variety of different syntheses and are often biologically active. In addition, they function as protecting groups, oxidizing agents, ligands in metal complexes and specific catalysts.
The chemical oxygenation of pyridine, derivatives thereof and other N-heterocycles is relatively complex, requires aggressive/toxic chemicals/catalysts and leads to a series of undesired by-products (e.g. 2-, 3- and/or 4-hydroxypyridine derivatives) and low isomer yields. According to the literature, pyridine N-oxide can be chemically synthesized from pyridine using the following starting compounds among others:                hydrogen peroxide (30%), acetic acid and pyridine (80° C. in pyridine/water)        phosphotungstic acid on silicon dioxide and pyridine (80° C. in pyridine)        tungstic acid salts, hydrogen peroxide (30%) and pyridine (80° C. in pyridine)        organic hydrotrioxides and pyridine (−80 to −60° C. in pyridine)        hydrogen peroxide, manganese tetrakis(2,6-chlorophenyl)porphyrin (25° C. in dichloroethane)        dimethyloxirane and pyridine (0° C., in dichloroethane)        perfluoro(cis-2,3-dialkyloxaziridine) and pyridine (25° C. in pyridine).        
Oxygenation reactions on heterocyclic nitrogen atoms are usually based on generation, in the presence of electron donors and molecular oxygen (O2) or a peroxide/trioxide (R—OOH, R—OOOH), by a catalyst, of a reactive oxygen species which attacks the nitrogen directly. These highly reactive oxygen species have only limited regioselectivity. For this reason, the yields in chemical N-oxygenations are low, and they lead to undesired by-products and require a complicated operation.
It is known that an intracellular enzyme, methane monooxygenase (MMO, EC 14.13.25), converts pyridine to pyridine N-oxide in an unspecific side reaction. The MMO enzyme consists of several protein components and is formed by methylotrophic bacteria (e.g. Methylococcus capsulatus); it requires complex electron donors such as NADH or NADPH, auxiliary proteins (flavin reductases, regulator protein) and molecular oxygen (O2). The natural substrate of MMO is methane, which is oxidized to methanol.
As a particularly unspecific biocatalyst, MMO oxygenates/hydroxylates, as well as methane, a series of further substrates such as n-alkanes and their derivatives, cycloalkanes, aromatics, carbon monoxide and heterocycles. The latter and pyridine in particular are, however, converted only with very low rates; the specific activity with respect to pyridine is 0.029 unit mg−1 of protein (Colby et al. 1977: The soluble methane monooxygenase of Methylococcus capsulatus. Biochem. J. 165: 395-402). Utilization of the enzyme in biotechnology is currently not possible, since it is difficult to isolate, like most intracellular enzymes, it is of low stability, and the cosubstrates required are relatively expensive.
Pyridine-degrading bacteria such as Rhodococcus spp. or Arthrobacter spp. do not possess any enzyme which generates pyridine N-oxide, but rather utilize enzymes which hydroxylate the pyridine ring at the carbon (rare) or reduce particular bonds of the ring (common) and thus initiate the degradation (Fetzner, S., 1998: Bacterial degradation of pyridine, indole, quinoline, and their derivatives under different redox conditions. Appl. Microbiol. Biotechnol. 49: 237-250).